Moss spores germinate to form an alga-like filamentous structure called the protonemata. Protonemata are the earliest stage (the haploid phase) of a bryophyte life cycle and eventually give rise to a mature gametophyte. Protonemata of the moss Physcomitrella patens (P. patens) are important not only in their life cycle, but also for research. Protonemata are used for various things such as RNA/DNA extractions and protoplast isolation. We can obtain high yield of intact protoplasts from protonemata. Protoplasts can be used to study a variety of cellular processes, such as subcellular localization of proteins, isolation and analyses of intact organelles and DNA transformation. In addition, the completed sequence of the P. patens genome facilitates the use of genetic and molecular approaches to identify genes and the ability of the moss to undergo homologous recombination at appreciable frequency offers a powerful way to determine gene function. Therefore, culture of P. patens protonemata is critical.

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Tissue Culturing and Harvesting of Protonemata from the Moss Physcomitrella patens

[Abstract]
Moss spores germinate to form an alga-like filamentous structure called the protonemata. Protonemata are the earliest stage (the haploid phase) of a bryophyte life cycle and eventually give rise to a mature gametophyte. Protonemata of the moss Physcomitrella patens (P. patens) are important not only in their life cycle, but also for research. Protonemata are used for various things such as RNA/DNA extractions and protoplast isolation. We can obtain high yield of intact protoplasts from protonemata. Protoplasts can be used to study a variety of cellular processes, such as subcellular localization of proteins, isolation and analyses of intact organelles and DNA transformation. In addition, the completed sequence of the P. patens genome facilitates the use of genetic and molecular approaches to identify genes and the ability of the moss to undergo homologous recombination at appreciable frequency offers a powerful way to determine gene function. Therefore, culture of P. patens protonemata is critical.

Materials and Reagents

A vigorously growing tissue which is about 10 d old (the earliest stage of gametophyte; Physcomitrella patens subspecies patens (Gransden) was used as the tissue and it was obtained from Ralph S. Quatrano (Department of Biology, Washington University in St. Louis, MO 63130, USA)

200 ml sterile distilled water

70% alcohol in a spray bottle (for surface sterilization)

Growth medium (see Recipes)

Equipment

1 L flask in which to prepare the growth medium

Sterile petri dishes (90-mm)

Sterile cellophane discs

Sterile tweezers

Sterile test tubes (25 x 150 mm)

Micropore surgical tape

Sterile pipettes (1 ml)

Sterile tips (1 ml)

Dispensing instrument (e.g., IKA T 10 basic ULTRA-TURRAX®)

Laminar flow cabinet

Autoclave (e.g., Sanyo, model: MLS-3780)

Procedure

Note: Steps 2-12 should be carried out under sterile conditions.

BCDA medium is prepared according to Table 1 and sterilized by an autoclave for 20 min at 121 °C;

Surface sterilization is performed with 70% alcohol in the Laminar flow hood;

Media was cooled to 60 °C and approximately 30 ml BCDA medium was poured into sterile petri dishes (90 mm). This was allowed to cool until the media solidified; Place a piece of sterile cellophane discs onto the BCDA medium (Video 1);

Video 1. Place a piece of sterile cellophane discs onto the BCDA medium

To play the video, you need to install a newer version of Adobe Flash Player.

To play the video, you need to install a newer version of Adobe Flash Player.

Seal the Petri dishes with micropore surgical tape;

Incubate these fragments for 6-7 d under standard conditions (25 °C) with a light cycle of 16 h of light/8 h of darkness and a light intensity of 70-80 μmol/s/m2;

Transfer 1-2 dishes of the tissue (over cellophane) onto BCD solid media (Table 2) growth 3-4 d under the same culture conditions to step 9 for next cycle (this is a vigorously growing tissue which is about 10 d old tissues; Video 4);

Video 4. Transfer 1-2 dishes of the tissue onto BCD solid media growth 3-4 d under the same culture conditions to step 9 for next cycle

To play the video, you need to install a newer version of Adobe Flash Player.

Harvest the tissue (protonemata of P. patens from step 9) for downstream analysis by scraping them from the cellophane using sterile tweezers.

For tissue stock, take a tiny amount (about 2 mm2; using sterile tweezers) of 10 d old tissues into sterile test tubes with BCD medium. Wrap the tubes in foil and store them at 4 °C. They remain viable for about six months.

Recipes

Growth medium

Table 1. BCDA medium recipe

Reagent

Quantity (for 1 L)

Final concentration

Solution B

10 ml

1 mM MgSO4

Solution C

10 ml

1.84 mM KH2PO4

Solution D

10 ml

10 mM KNO3

CaCl2

111 mg

1 mM

FeSO4.7H2O

12.5 mg

45 μM

(NH4)2C4H4O6

0.92 g

5 mM

Agar

7.5 g

0.75% (w/v)

Glucose

5 g

0.5% (w/v)

Hoagland’s A-Z trace

1 ml

Trace element solution

H2O

To 1 L

Table 2. BCD medium recipe

Reagent

Quantity (for 1 L)

Final concentration

Solution B

10 ml

1 mM MgSO4

Solution C

10 ml

1.84 mM KH2PO4

Solution D

10 ml

10 mM KNO3

CaCl2

111 mg

1 mM

FeSO4.7H2O

12.5 mg

45 μM

Agar

7.5 g

0.75% (w/v)

Glucose

5 g

0.5% (w/v)

Hoagland’s A-Z trace

1 ml

Trace element solution

H2O

To 1 L

Table 3. Recipe of Solution B, C, D and Hoagland’s A-Z trace

Reagent

Quantity (for 1 L)

Final concentration

Solution B

MgSO4.7H2O

25 g

0.1 M

H2O

To 1 L

Solution C

KH2PO4

25 g

184 mM

H2O

To 1 L

Adjust the pH to 6.5 using KOH

Solution D

KNO3

101 g

1 M

H2O

To 1 L

Hoagland’s A-Z trace

Al2(SO4)3.K2SO4.24H2O

55 mg

0.006% (w/v)

CoCl2.6H2O

55 mg

0.006% (w/v)

CuSO4.5H2O

55 mg

0.006% (w/v)

H3BO3

614 mg

0.061% (w/v)

KBr

28 mg

0.003% (w/v)

KI

28 mg

0.003% (w/v)

LiCl

28 mg

0.003% (w/v)

MnCl2.4H2O

389 mg

0.039% (w/v)

SnCl2.2H2O

28 mg

0.003% (w/v)

ZnSO4.7H2O

55 mg

0.006% (w/v)

H2O

To 1 L

Solution B, C, D and Hoagland’s A-Z trace are sterilized and then stored at 4 °C or room temperature.

Acknowledgments

This work was supported by grants from Beijing Natural Science Foundation (No. 5132004), China Postdoctoral Science Foundation and State Education Ministry Scientific Research Foundation for the Returned Overseas Chinese Scholars to Dr. Wang.

How to cite:Wang, X. and He, Y. (2015). Tissue Culturing and Harvesting of Protonemata from the Moss Physcomitrella patens. Bio-protocol 5(15): e1556. DOI: 10.21769/BioProtoc.1556; Full Text

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